Cancer cells produce substances in a large amount which are produced in a small amount or are rarely produced in normal cells. Cancer cells can be detected by detecting or quantitatively determining such substances. The substances which are produced much more in cancer cells than in normal cells include oncogene products, growth factors and the like, and some of them related to malignant transformation, growth, progress, metastasis and fixation of the cells. Cancer can be diagnosed by detecting or quantitatively determining those substances which are characteristic to cancer cells, that is, the so-called tumor markers.
Up to now, tumor markers which have been used include embryonal cancer antigens such as carcinoembryonic antigen (CEA), α-fetoprotein (AFP) and CA 125; enzymes such as nerve-specific enolase (NSE), acidic phosphatase and creatine kinase (CK); and hormone-related substances such as adrenocorticotropic hormone (ACTED, anti-diuretic hormone (ADH) and calcitonin (CT). In colorectal cancer, CEA, CA19-9, NCC-ST-439, STN and the like have been used as markers for determining the therapeutic effect and recurrence. However, when the above-described tumor markers are used, there are many cases where even malignant tumor such as cancer is determined to be negative and, even in healthy persons and patients with benign tumor, there are some cases where they are determined to be pseudo-positive. For example, when the cases where tumor marker is positive were checked for diseased period in colorectal cancer, CEA, CA19-9, NCC-ST-439 and STN were detected only in 36%, 30%, 35% and 21%, respectively, of the patients during a stage where healing excision is possible whereby those tumor markers are not sufficient tumor markers for finding of colorectal cancer in early stages [Tumor Markers for Colorectal Cancer, CRC, 1(4), 42 (1992)].
In order to enhance sensitivity and specificity in diagnosis of cancer, it is effective to combine plural tumor markers. When a new tumor marker is found, it is possible to enhance sensitivity and precision of diagnosis of cancer by a sole use of a new tumor marker or by a combined use with the conventional tumor marker.
In pancreatic cancer, general clinical test items show normal value and, in addition, no characteristic clinical observation is available in early stage of the disease and, accordingly, it is difficult to find patients suffering from pancreatic cancer in early stages. In patients suffering from pancreatic cancer where biliary obstruction or liver metastasis happens, there are some cases where alkaline phosphatase value and bilirubin value increase. In cancer of pancreas, pancreatitis is generated in a peripheral side of obstructed pancreatic duct due to tumor and, as a result, enzymes secreted outside the pancreas such as amylase, elastase and RNase and inhibitors for the enzymes come into blood and increase whereby the enzymes and the inhibitors for the enzymes as such are used as tumor markers and, for example, PSTI pancreatic secretory trypsin inhibitor) has been known. PSTI is an inhibitor for trypsin secreted into pancreatic juice and PSTI in blood highly increases in various kinds of malignant tumor patients. It is noted in high frequency particularly in patients suffering from pancreatic cancer [Rinsho Byori, 11, 1229 (1986)].
CA19-9 has been widely used as a tumor marker for diagnosis and treatment monitor of pancreatic duct cancer having a high expressing frequency. As other tumor markers for pancreatic duct cancer, CEA, SLX, NCC-ST-439, sialyl Tn, DuPan-2, ferritin and the like have been known. However, in primary pancreatic cancer without metastasis, there are many cases where measured values of those tumor markers do not increase and there are also many cases where the judgment is pseudo-positive from the measured values of those tumor markers whereupon no well-reliable diagnostic method for pancreatic cancer has been known.
In the most precise and the highest cost effective method in diagnosis and staging of pancreatic cancer, CT (computed tomography) is carried out in the initial test. When it is found to be impossible to extirpate tissues or to be metastasized by CT, percutaneous suction with a needle is carried out for tissue diagnosis. If excisable tumor is found or no tumor is found at all by CT, an ultrasonic endoscope is used. Besides that, ultrasonic wave and endoscope retrograde cholangiopanreatography are used for a common test. It is rare to carry out arteriography and pancreatic function test in order to determine whether excision is possible. Furthermore, when diagnosis is difficult, exploratory laparotomy may be carried out.
However, since pancreas is a retroperitoneal organ, it is not easy to precisely find pancreatic cancer in early stages by those diagnostic methods. An early detection contributes in improvement in curing rate and, therefore, there has been a demand for an excellent diagnostic method for pancreatic cancer in an early stage.
The DNA sequence of PERP (also referred to as “THW” or “PIGPC1”) is known (WO98/55508, WO99/54461, WO00/55350, WO01/22920, WO01/66719, WO00/61612, WO02/00174, WO02/47534, US2003-0064947, US2003-0065157, WO00/55629, WO02/60317, US2002-0119463).
A polypeptide encoded by the PERP gene is a protein consisting of 193 amino acids and is presumed to be a four-times transmembrane protein from its primary sequence. It has been known that a polypeptide encoded by PERP gene is a protein concerning p53-dependent apoptosis [Genes & Development 14, 704 (2000)]. It has been further shown that, in thymus cells and nerve cells prepared from PERP gene knocked out mice, apoptosis induction upon DNA damage is partially inhibited [Curr. Biol., 13, 1985 (2003)]. It has been also reported that PERP is a gene in which expression is lowered in highly metastatic cancer cells [Anticancer Research, 20, 2801 (2000)].
As an antibody binding to a polypeptide encoded by the PERP gene (hereinafter referred to as “anti-PERP antibody”), a polyclonal antibody prepared from an intracellular partial peptide in the C terminal or a partial peptide of the first extracellular loop in a PERP gene product as an immunogen has been known (Home page of Pro Sci Incorporated, on line, retrieved on Mar. 31, 2004, internet <www.prosci-inc.com/Antibody-TDS/2451%20PERP.html>, home page of Novus Biologicals, Inc., on line, retrieved on Mar. 31, 2004, internet <www.novus-biologicals.com/print_data_sheet.php/4000>). These polyclonal antibodies have been shown to be applicable to Western blotting or immunohistostaining. Up to now, no antibody which recognizes the three-dimensional structure of an extracellular region of polypeptide encoded by PERP gene and binds to the extracellular region has been known.
It has been known that, when an antibody of non-human animals such as a mouse antibody is administered to human, it is usually recognized as a xenobiotic substance and accordingly that a human antibody against a mouse antibody (human anti-mouse antibody: HAMA) is induced in human body. It has been known that HAMA reacts with the administered mouse antibody to induce side effects [J. Clin. Oncol., 2, 881 (1984), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80, 932 (1988), Proc. Natl. Acad. Sci. USA 82, 1242 (1985)], promotes the disappearance of the mouse antibody from the body [Blood 65, 1349 (1985), J. Nuc. Med., 26, 1011 (1985), J. Natl. Cancer Inst., 80, 937 (1988)] and reduces the therapeutic effect of the mouse antibody [J. Immunol., 135, 1530 (1985), Cancer Res., 46, 6489 (1986)].
In order to solve these problems, it has been attempted to prepare a humanized antibody such as a human chimeric antibody or a human CDR-grafted antibody from an antibody of non-human animals by using genetic recombination techniques.
In comparison with an antibody of non-human animals such as a mouse antibody, the humanized antibody has various advantages in clinical application to human. It has been reported, for example, that, in experiments using monkeys, immunogenicity is lowered and its half-life period in blood becomes longer in comparison with a mouse antibody [Cancer Res., 56, 1118 (1996), Immunol., 85, 668 (1995)]. Thus it is expected that, in comparison with the antibody of non-human animals, the humanized antibody has little side effects in human and its therapeutic effect lasts for a long period.
In addition, since the humanized antibody is prepared by using genetic recombination techniques, it can be prepared as molecules in various forms. For example, when the γ1 subclass is used as a heavy chain (hereinafter referred to as “H chain”) constant region (hereinafter referred to as “C region”) (H chain C region will be referred to as “CH”) of a human antibody, it is possible to prepare a humanized antibody having a high effector function such as antibody-dependent cellular cytotoxicity (hereinafter referred to as “ADCC”) [Cancer Res., 56, 1118 (1996)] and imposed half-life in blood can be expected in comparison with a mouse antibody [Immunol., 85, 668 (1995)]. Particularly, in the treatment where expressed cell numbers of polypeptide encoded by the PERP gene are lowered high cytotoxic activity such as complement-dependent cytotoxic activity (hereinafter referred to as “CDC activity”) and ADCC activity via Fc region of an antibody (region which is other than a hinge region of the antibody heavy chain) is important to the therapeutic effect and, therefore, the humanized antibody is preferred in comparison with the antibody of non-human animals such as a mouse antibody [J. Immunol., 144, 1382 (1990), Nature, 322, 323 (1988)].
Moreover, as a result of the progress in protein engineering and genetic engineering in recent years, the humanized antibody can also be prepared as antibody fragment having a low molecular weight such as Fab, Fab′, F(ab′)2, a single chain antibody (hereinafter referred to as “scFv”) [Science, 242, 423 (1988)], a dimerized V region fragment (hereinafter be referred to as “diabody”) [Nature Biotechnol, 15, 629 (1997)], a disulfide stabilized V region fragment (hereinafter referred to as “dsFv”) [Molecular Immunol, 32, 249 (1995)], a peptide comprising CDR [J. Biol. Chem., 271, 2966 (1996)] and the like, and these antibody fragments are better in transition to target tissues than whole antibody molecules [Cancer Res., 52, 3402 (1992)].
The above-described facts show that, as an antibody to be used for clinical application to human, a humanized antibody or the antibody fragment thereof is preferred than an antibody of non-human animals such as a mouse antibody.